RNA Interference Mediating Small RNA Molecules

ABSTRACT

Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a  Drosophila  in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of Ser. No. 10/433,050 filed Jul. 26,2004, which is a 35 USC § 371 National Phase Entry Application fromPCT/EP01/13968, filed Nov. 29, 2001, and designating the U.S., whichclaims the benefit of provisional application No. 60/279,661 filed Mar.30, 2001, and European Application No. 00126325.0 filed Dec. 1, 2000.All of these applications are incorporated herein by reference.

DESCRIPTION

The present invention relates to sequence and structural features ofdouble-stranded (ds) RNA molecules required to mediate target-specificnucleic acid modifications such as RNA-interference and/or DNAmethylation.

The term “RNA interference” (RNAi) was coined after the discovery thatinjection of dsRNA into the nematode C. elegans leads to specificsilencing of genes highly homologous in sequence to the delivered dsRNA(Fire et al., 1998). RNAi was subsequently also observed in insects,frogs (Oelgeschlager et al., 2000), and other animals including mice(Svoboda et al., 2000; Wianny and Zernicka-Goetz, 2000) and is likely toalso exist in human. RNAi is closely linked to the post-transcriptionalgene-silencing (PTGS) mechanism of co-suppression in plants and quellingin fungi (Catalanotto et al., 2000; Cogoni and Macino, 1999; Dalmay etal., 2000; Ketting and Plasterk, 2000; Mourrain et al., 2000; Smardon etal., 2000) and some components of the RNAi machinery are also necessaryfor post-transcriptional silencing by co-suppression (Catalanotto etal., 2000; Dernburg et al., 2000; Ketting and Plasterk, 2000). The topichas also been reviewed recently (Bass, 2000; Bosher and Labouesse, 2000;Fire, 1999; Plasterk and Ketting, 2000; Sharp, 1999; Sijen and Kooter,2000), see also the entire issue of Plant Molecular Biology, vol. 43,issue 2/3, (2000).

In plants, in addition to PTGS, introduced transgenes can also lead totranscriptional gene silencing via RNA-directed DNA methylation ofcytosines (see references in Wassenegger, 2000). Genomic targets asshort as 30 bp are methylated in plants in an RNA-directed manner(Pelissier, 2000). DNA methylation is also present in mammals.

The natural function of RNAi and co-suppression appears to be protectionof the genome against invasion by mobile genetic elements such asretrotransposons and viruses which produce aberrant RNA or dsRNA in thehost cell when they become active (Jensen et al, 1999; Ketting et al.,1999; Ratcliff et al., 1999; Tabara et al., 1999). Specific mRNAdegradation prevents transposon and virus replication although someviruses are able to overcome or prevent this process by expressingproteins that suppress PTGS (Lucy et al., 2000; Voinnet et al., 2000).

DsRNA triggers the specific degradation of homologous RNAs only withinthe region of identity with the dsRNA (Zamore et al., 2000). The dsRNAis processed to 21-23 nt RNA fragments and the target RNA cleavage sitesare regularly spaced 21-23 nt apart. It has therefore been suggestedthat the 21-23 nt fragments are the guide RNAs for target recognition(Zamore et al., 2000). These short RNAs were also detected in extractsprepared from D. melanogaster Schneider 2 cells which were transfectedwith dsRNA prior to cell lysis (Hammond et al. 2000), however, thefractions that displayed sequence-specific nuclease activity alsocontained a large fraction of residual dsRNA. The role of the 21-23-ntfragments in guiding mRNA cleavage is further supported by theobservation that 21-23 nt fragments isolated from processed dsRNA areable, to some extent, to mediate specific mRNA degradation (Zamore etal., 2000). RNA molecules of similar size also accumulate in planttissue that exhibits PTGS (Hamilton and Baulcombe, 1999).

Here, we use the established Drosophila in vitro system (Tuschl et al.,1999; Zamore et al., 2000) to further explore the mechanism of RNAi. Wedemonstrate that short 21 and 22 nt RNAs, when base-paired with 3′overhanging ends, act as the guide RNAs for sequence-specific mRNAdegradation. Short 30 bp dsRNAs are unable to mediate RNAi in thissystem because they are no longer processed to 21 and 22 nt RNAs.Furthermore, we defined the target RNA cleavage sites relative to the 21and 22 nt short interfering RNAs (siRNAs) and provide evidence that thedirection of dsRNA processing determines whether a sense or an antisensetarget RNA can be cleaved by the produced siRNP endonuclease complex.Further, the siRNAs may also be important tools for transcriptionalmodulating, e.g. silencing of mammalian genes by guiding DNAmethylation.

Further experiments in human in vivo cell culture systems (HeLa cells)show that double-stranded RNA molecules having a length of preferablyfrom 19-25 nucleotides have RNAi activity. Thus, in contrast to theresults from Drosophila also 24 and 25 nt long double-stranded RNAmolecules are efficient for RNAi.

The object underlying the present invention is to provide novel agentscapable of mediating target-specific RNA interference or othertarget-specific nucleic acid modifications such as DNA methylation, saidagents having an improved efficacy and safety compared to prior artagents.

The solution of this problem is provided by an isolated double-strandedRNA molecule, wherein each RNA strand has a length from 19-25,particularly from 19-23 nucleotides, wherein said RNA molecule iscapable of mediating target-specific nucleic acid modifications,particularly RNA interference and/or DNA methylation. Preferably atleast one strand has a 3′-overhang from 1-5 nucleotides, more preferablyfrom 1-3 nucleotides and most preferably 2 nucleotides. The other strandmay be blunt-ended or has Up to 6 nucleotides 3′ overhang. Also, if bothstrands of the dsRNA are exactly 21 or 22 nt, it is possible to observesome RNA interference when both ends are blunt (0 nt overhang). The RNAmolecule is preferably a synthetic RNA molecule which is substantiallyfree from contaminants occurring in cell extracts, e.g. from Drosophilaembryos. Further, the RNA molecule is preferably substantially free fromany non-target-specific contaminants, particularly non-target-specificRNA molecules e.g. from contaminants occurring in cell extracts.

Further, the invention relates to the use of isolated double-strandedRNA molecules, wherein each RNA strand has a length from 19-25nucleotides, for mediating, target-specific nucleic acid modifications,particularly RNAi, in mammalian cells, particularly in human cells.

Surprisingly, it was found that synthetic short double-stranded RNAmolecules particularly with overhanging 3′-ends are sequence-specificmediators of RNAi and mediate efficient target-RNA cleavage, wherein thecleavage site is located near the center of the region spanned by theguiding short RNA.

Preferably, each strand of the RNA molecule has a length from 20-22nucleotides (or 20-25 nucleotides in mammalian cells), wherein thelength of each strand may be the same or different. Preferably, thelength of the 3′-overhang reaches from 1-3 nucleotides, wherein thelength of the over-hang may be the same or different for each strand.The RNA-strands preferably have 3′-hydroxyl groups. The 5′-terminuspreferably comprises a phosphate, diphosphate, triphosphate or hydroxylgroup. The most effective dsRNAs are composed of two 21 nt strands whichare paired such that 1-3, particularly 2 nt 3′ overhangs are present onboth ends of the dsRNA.

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. However, not all positions of a siRNA contribute equally totarget recognition. Mismatches in the center of the siRNA duplex aremost critical and essentially abolish target RNA cleavage. In contrast,the 3′ nucleotide of the siRNA strand (e.g. position 21) that iscomplementary to the single-stranded target RNA, does not contribute tospecificity of the target recognition. Further, the sequence of theunpaired 2-nt 3′ overhang of the siRNA strand with the same polarity asthe target RNA is not critical for target RNA cleavage as only theantisense siRNA strand guides target recognition. Thus, from thesingle-stranded overhanging nucleotides only the penultimate position ofthe antisense siRNA (e.g. position 20) needs to match the targeted sensemRNA.

Surprisingly, the double-stranded RNA molecules of the present inventionexhibit a high in vivo stability in serum or in growth medium for cellcultures. In order to further enhance the stability, the 3′-overhangsmay be stabilized against degradation, e.g. they may be selected suchthat they consist of purine nucleotides, particularly adenosine orguanosine nucleotides. Alternatively, substitution of pyrimidinenucleotides by modified analogues, e.g. substitution of uridine 2 nt 3′overhangs by 2′-deoxythymidine is tolerated and does not affect theefficiency of RNA interference. The absence of a 2′ hydroxylsignificantly enhances the nuclease resistance of the overhang in tissueculture medium.

In an especially preferred embodiment of the present invention the RNAmolecule may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific activity, e.g. the RNAi mediating activity is notsubstantially effected, e.g. in a region at the 5′-end and/or the 3′-endof the double-stranded RNA molecule. Particularly, the overhangs may bestabilized by incorporating modified nucleotide analogues.

Preferred nucleotide analogues are selected from sugar- orbackbone-modified ribonucleotides. It should be noted, however, thatalso nucleobase-modified ribonucleotides, i.e. ribonucleotides,containing a non-naturally occurring nucleobase instead of a naturallyoccurring nucleobase such as uridines or cytidines modified at the5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosinesand guanosines modified at the 8-position, e.g. 8-bromo guanosine; deazanucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides,e.g. N6-methyl adenosine are suitable. In preferred sugar-modifiedribonucleotides the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I. In preferredbackbone-modified ribonucleotides the phosphoester group connecting toadjacent ribonucleotides is replaced by a modified group, e.g. ofphosphothioate group. It should be noted that the above modificationsmay be combined.

The sequence of the double-stranded RNA molecule of the presentinvention has to have a sufficient identity to a nucleic acid targetmolecule in order to mediate target-specific RNAi and/or DNAmethylation. Preferably, the sequence has an identity of at least 50%,particularly of at least 70% to the desired target molecule in thedouble-stranded portion of the RNA molecule. More preferably, theidentity is at least 85% and most preferably 100% in the double-strandedportion of the RNA molecule. The identity of a double-stranded RNAmolecule to a predetermined nucleic acid target molecule, e.g. an mRNAtarget molecule may be determined as follows:

$I = {\frac{n}{L} \times 100}$

wherein I is the identity in percent, n is the number of identicalnucleotides in the double-stranded portion of the ds RNA and the targetand L is the length of the sequence overlap of the double-strandedportion of the dsRNA and the target.

Alternatively, the identity of the double-stranded RNA molecule to thetarget sequence may also be defined including the 3′ overhang,particularly an overhang having a length from 1-3 nucleotides. In thiscase the sequence identity is preferably at least 50%, more preferablyat least 70% and most preferably at least 85% to the target sequence.For example, the nucleotides from the 3′ overhang and up to 2nucleotides from the 5′ and/or 3′ terminus of the double strand may bemodified without significant loss of activity.

The double-stranded RNA molecule of the invention may be prepared by amethod comprising the steps:

-   (a) synthesizing two RNA strands each having a length from 19-25,    e.g. from 19-23 nucleotides, wherein said RNA strands are capable of    forming a double-stranded RNA molecule, wherein preferably at least    one strand has a 3′-overhang from 1-5 nucleotides,-   (b) combining the synthesized RNA strands under conditions, wherein    a double-stranded RNA molecule is formed, which is capable of    mediating target-specific nucleic acid modifications, particularly.    RNA interference and/or DNA methylation.

Methods of synthesizing RNA molecules are known in the art. In thiscontext, it is particularly referred to chemical synthesis methods asdescribed in Verma and Eckstein (1998).

The single-stranded RNAs can also be prepared by enzymatic transcriptionfrom synthetic DNA templates or from DNA plasmids isolated fromrecombinant bacteria. Typically, phage RNA polymerases are used such asT7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck (1989)).

A further aspect of the present invention relates to a method ofmediating target-specific nucleic acid modifications, particularly RNAinterference and/or DNA methylation in a cell or an organism comprisingthe steps:

-   (a) contacting the cell or organism with the double-stranded RNA    molecule of the invention under conditions wherein target-specific    nucleic acid modifications may occur and-   (b)-mediating a target-specific nucleic acid modification effected    by the double-stranded RNA towards a target nucleic acid having a    sequence portion substantially corresponding to the double-stranded    RNA.

Preferably the contacting step (a) comprises introducing thedouble-stranded RNA molecule into a target cell, e.g. an isolated targetcell, e.g. in cell culture, a unicellular microorganism or a target cellor a plurality of target cells within a multicellular organism. Morepreferably, the introducing step comprises a carrier-mediated delivery,e.g. by liposomal carriers or by injection.

The method of the invention may be used for determining the function ofa gene in a cell or an organism or even for modulating the function of agene in a cell or an organism, being capable of mediating RNAinterference. The cell is preferably a eukaryotic cell or a cell line,e.g. a plant cell or an animal cell, such as a mammalian cell, e.g. anembryonic cell, a pluripotent stem cell, a tumor cell, e.g. ateratocarcinoma cell or a virus-infected cell. The organism ispreferably a eukaryotic organism, e.g. a plant or an animal, such as amammal, particularly a human.

The target gene to which the RNA molecule of the invention is directedmay be associated with a pathological condition. For example, the genemay be a pathogen-associated gene, e.g. a viral gene, a tumor-associatedgene or an autoimmune disease-associated gene. The target gene may alsobe a heterologous gene expressed in a recombinant cell or a geneticallyaltered organism. By determinating or modulating, particularly,inhibiting the function of such a gene valuable information andtherapeutic benefits in the agricultural field or in the medicine orveterinary medicine field may be obtained.

The dsRNA is usually administered as a pharmaceutical composition. Theadministration may be carried out by known methods, wherein a nucleicacid is introduced into a desired target cell in vitro or in vivo.Commonly used gene transfer techniques include calcium phosphate,DEAE-dextran, electroporation and microinjection and viral methods(Graham, F. L. and van der Eb, A. J. (1973) Virol; 52, 456; McCutchan,J. H. and Pagano, J. S. (1968), J. Natl. Cancer Inst. 41, 351; Chu, G.et al (1987), Nucl. Acids Res. 15, 1311; Fraley, R. et al. (1980), J.Biol. Chem. 255, 10431; Capecchi, M. R. (1980), Cell 22, 479). A recentaddition to this arsenal of techniques for the introduction of DNA intocells is the use of cationic liposomes (Felgner, P. L. et al. (1987),Proc. Natl. Acad. Sci. USA 84, 7413). Commercially available cationiclipid formulations are e.g. Tfx 50 (Promega) or Lipofectamin2000 (LifeTechnologies).

Thus, the invention also relates to a pharmaceutical compositioncontaining as an active agent at least one double-stranded RNA moleculeas described above and a pharmaceutical carrier. The composition may beused for diagnostic and for therapeutic applications in human medicineor in veterinary medicine.

For diagnostic or therapeutic applications, the composition may be inform of a solution, e.g. an injectable solution, a cream, ointment,tablet, suspension or the like. The composition may be administered inany suitable way, e.g. by injection, by oral, topical, nasal, rectalapplication etc. The carrier may be any suitable pharmaceutical carrier.Preferably, a carrier is used, which is capable of increasing theefficacy of the RNA molecules to enter the target-cells. Suitableexamples of such carriers are liposomes, particularly cationicliposomes. A further preferred administration method is injection.

A further preferred application of the RNAi method is a functionalanalysis of eukaryotic cells, or eukaryotic non-human organisms,preferably mammalian cells or organisms and most preferably human cells,e.g. cell lines such as HeLa or 293 or rodents, e.g. rats and mice. Bytransfection with suitable double-stranded RNA molecules which arehomologous to a predetermined target gene or DNA molecules encoding asuitable double-stranded RNA molecule a specific knockout phenotype canbe obtained in a target cell, e.g. in cell culture or in a targetorganism. Surprisingly it was found that the presence of shortdouble-stranded RNA molecules does not result in an interferon responsefrom the host cell or host organism.

Thus, a further subject matter of the invention is a eukaryotic cell ora eukaryotic non-human organism exhibiting a target gene-specificknockout phenotype comprising an at least partially deficient expressionof at least one endogeneous target-gene wherein said cell or organism istransfected with at least one double-stranded RNA molecule capable ofinhibiting the expression of at least one endogeneous target gene orwith a DNA encoding at least one double stranded RNA molecule capable ofinhibiting the expression of at least one endogeneous target gene. Itshould be noted that the present invention allows a target-specificknockout of several different endogeneous genes due to the specificityof RNAi.

Gene-specific knockout phenotypes of cells or non-human organisms,particularly of human cells or non-human mammals may be used in analyticprocedures, e.g. in the functional and/or phenotypical analysis ofcomplex physiological processes such as analysis of gene expressionprofiles and/or proteomes. For example, one may prepare the knock-outphenotypes of human genes in cultured cells which are assumed to beregulators of alternative splicing processes. Among these genes areparticularly the members of the SR splicing factor family, e.g. ASF/SF2,SC35, SRp20, SRp40 or SRp55. Further, the effect of SR proteins on themRNA profiles of predetermined alternatively spliced genes such as CD44may be analysed. Preferably the analysis is carried out byhigh-throughput methods using oligonucteotide based chips.

Using RNAi based knockout technologies, the expression of an endogeneoustarget gene may be inhibited in a target cell or a target organism.

The endogeneous gene may be complemented by an exogeneous target nucleicacid coding for the target protein or a variant or mutated form of thetarget protein, e.g. a gene or a cDNA, which may optionally be fused toa further nucleic acid sequence encoding a detectable peptide orpolypeptide, e.g. an affinity tag, particularly a multiple affinity tag.Variants or mutated forms of the target gene differ from the endogeneoustarget gene in that they encode a gene product which differs from theendogeneous gene product on the amino acid-level by substitutions,insertions and/or deletions of single or multiple amino acids. Thevariants or mutated forms may have the same biological activity as theendogeneous target gene. On the other hand, the variant or mutatedtarget gene may also have a biological activity, which differs from thebiological activity of the endogeneous target gene, e.g. a partiallydeleted activity, a completely deleted activity, an enhanced activityetc.

The complementation may be accomplished by coexpressing the polypeptideencoded by the exogeneous nucleic acid, e.g. a fusion protein comprisingthe target protein and the affinity tag and the double stranded RNAmolecule for knocking out the endogeneous gene in the target cell. Thiscoexpression may be accomplished by using a suitable expression vectorexpressing both the polypeptide encoded by the exogeneous nucleic acid,e.g. the tag-modified target protein and the double stranded RNAmolecule or alternatively by using a combination of expression vectors.Proteins and protein complexes which are synthesized de novo in thetarget cell will contain the exogeneous gene product, e.g. the modifiedfusion protein. In order to avoid suppression of the exogeneous geneproduct expression by the RNAi duplex molecule, the nucleotide sequenceencoding the exogeneous nucleic acid may be altered on the DNA level(with or without cau-, sing mutations on the amino acid level) in thepart of the sequence which is homologous to the double stranded RNAmolecule. Alternatively, the endogeneous target gene may be complementedby corresponding nucleotide sequences from other species, e.g. frommouse.

Preferred applications for the cell or organism of the invention is theanalysis of gene expression profiles and/or proteomes. In an especiallypreferred embodiment an analysis of a variant or mutant form of one orseveral target proteins is carried out, wherein said variant or mutantforms are reintroduced into the cell or organism by an exogeneous targetnucleic acid as described above. The combination of knockout of anendogeneous gene and rescue by using mutated, e.g. partially deletedexogeneous target has advantages compared to the use of a knockout cell.Further, this method is particularly suitable for identifying functionaldomains of the target protein. In a further preferred embodiment acomparison, e.g. of gene expression profiles and/or proteomes and/orphenotypic characteristics of at least two cells or organisms is carriedout. These organisms are selected from:

-   (i) a control cell or control organism without target gene    inhibition,-   (ii) a cell or organism with target gene inhibition and-   (iii) a cell or organism with target gene inhibition plus target    gene complementation by an exogeneous target nucleic acid.

The method and cell of the invention are also suitable in a procedurefor identifying and/or characterizing pharmacological agents, e.g.identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents.

Thus, the present invention also relates to a system for identifyingand/or characterizing pharmacological agents acting on at least onetarget protein comprising:

-   (a) a eukaryotic cell or a eukaryotic non-human organism capable of    expressing at least one endogeneous target gene coding for said    target protein,-   (b) at least one double-stranded RNA molecule capable of inhibiting    the expression of said at least one endogeneous target gene, and-   (c) a test substance or a collection of test substances Wherein    pharmacological properties of said test substance or said collection    are to be identified and/or characterized.

Further, the system as described above preferably comprises:

-   (d) at least one exogeneous target nucleic acid coding for the    target protein or a variant or mutated form of the target protein    wherein said exogeneous target nucleic acid differs from the    endogeneous target gene on. the nucleic acid level such that the    expression of the exogeneous target nucleic acid is substantially    less inhibited by the double stranded RNA molecule than the    expression of the endogeneous target gene.

Furthermore, the RNA knockout complementation method may be used forpreparative purposes, e.g. for the affinity purification of proteins orprotein complexes from eukaryotic cells, particularly mammalian cellsand more particularly human cells. In this embodiment of the invention,the exogeneous target-nucleic acid preferably codes for a target proteinwhich is fused to an affinity tag.

The preparative method may be employed for the purification of highmolecular weight protein complexes which preferably have a mass of ≧150kD and more preferably of ≧500 kD and which optionally may containnucleic acids such as RNA. Specific examples are the heterotrimericprotein complex consisting of the 20 kD, 60 kD and 90 kD proteins of theU4/U6 snRNP particle, the splicing factor SF3b from the 17S U2 snRNPconsisting of 5 proteins having molecular weights of 14, 49, 120, 145and 0.155 kD and the 25S U4/U6/U5 tri-snRNP particle containing the U4,U5 and U6 snRNA molecules and about 30 proteins, which has a molecularweight of about 1.7 MD.

This method is suitable for functional proteome analysis in mammaliancells, particularly human cells.

Further, the present invention is explained in more detail in thefollowing figures and examples.

FIGURE LEGENDS

FIG. 1: Double-Stranded RNA as Short as 38 bp can Mediate RNai.

(A) Graphic representation of dsRNAs used for targeting Pp-luc mRNA.Three-series of blunt-ended dsRNAs covering a range of 29 to 504 bp wereprepared. The position of the first nucleotide of the sense strand ofthe dsRNA is indicated relative to the start codon of Pp-luc mRNA (p1).(B) RNA interference assay (Tuschl. et al., 1999). Ratios of targetPp-luc to control Rr-luc activity were normalized to a buffer control(black bar). DsRNAs (5 nM) were preincubated in Drosophila lysate for 15min at 25° C. prior to the addition of 7-methyl-guanosine-capped Pp-lucand Rr-luc mRNAs (˜50 pM). The incubation was continued for another hourand then analyzed by the dual luciferase assay (Promega). The data arethe average from at least four independent experiments ±standarddeviation.

FIG. 2: A 29 bp dsRNA is No Longer Processed to 21-23 nt Fragments.

Time course of 21-23 mer formation from processing of internally³²P-labeled dsRNAs (5 nM) in the Drosophila lysate. The length andsource of the dsRNA are indicated. An RNA-size marker (M) has beenloaded in the left lane and the fragment sizes are indicated. Doublebands at time zero are due to incompletely denatured dsRNA.

FIG. 3: Short dsRNAs Cleave the mRNA Target Only Once.

(A) Denaturing gel electrophoreses of the stable 5′ cleavage productsproduced by 1 h incubation of 10 nM sense or antisense RNA ³²P-labeledat the cap with 10 nM dsRNAs of the p133 series in Drosophila lysate.

Length markers were generated by partial nuclease T1 digestion andpartial alkaline hydrolysis (OH) of the cap-labeled target RNA. Theregions targeted by the dsRNAs are indicated as black bars on bothsides. The 20-23 nt spacing between the predominant cleavage sites forthe 111 bp long dsRNA is shown. The horizontal arrow indicatesunspecific cleavage not due to RNAi. (B) Position of the cleavage siteson sense and antisense target RNAs. The sequences of the capped 177 ntsense and 180 nt antisense target RNAs are represented in antiparallelorientation such that complementary sequence are opposing each other.The region targeted by the different dsRNAs are indicated by differentlycolored bars positioned between sense and antisense target sequences.Cleavage sites are indicated by circles: large circle for strongcleavage, small circle for weak cleavage. The ³²P-radiolabeled phosphategroup is marked by an asterisk.

FIG. 4: 21 and 22 nt RNA Fragments are Generated by an RNase III-LikeMechanism.

(A) Sequences of ˜21 nt RNAs after dsRNA processing. The ˜21 nt RNAfragments generated by dsRNA processing were directionally cloned andsequenced. Oligoribonucleotides originating from the sense strand of thedsRNA are indicated as blue lines, those originating from the antisensestrand as red lines. Thick bars are used if the same sequence waspresent in multiple clones, the number at the right indicating thefrequency. The target RNA cleavage sites mediated by the dsRNA areindicated as orange circles, large circle for strong cleavage, smallcircle for weak cleavage (see FIG. 3B). Circles on top of the sensestrand indicated cleavage sites within the sense target and circles atthe bottom of the dsRNA indicate cleavage site in the antisense target.Up to five additional nucleotides were identified in ˜21 nt fragmentsderived from the 3′ ends of the dsRNA. These nucleotides are randomcombinations of predominantly C, G, or A residues and were most likelyadded in an untemplated fashion during T7 transcription of thedsRNA-constituting strands. (B) Two-dimensional TLC analysis of thenucleotide composition of ˜21 nt RNAs. The ˜21 nt RNAs were generated byincubation of internally radiolabeled 504 bp Pp-luc dsRNA in Drosophilalysate, gel-purified, and then digested to mononucleotides with nucleaseP1 (top row) or ribonuclease T2 (bottom row). The dsRNA was internallyradiolabeled by transcription in the presence of one of the indicatedα-³²P nucleoside triphosphates. Radioactivity was detected byphosphorimaging. Nucleoside 5′-monophosphates, nucleoside3′-monophosphates, nucleoside 5′,3′-diphosphates, and inorganicphosphate are indicated as pN, Np, pNp, and p_(i), respectively. Blackcircles indicate UV-absorbing spots from non-radioactive carriernucleotides. The 3′,5′-bis-phosphates (red circles) were identified byco-migration with radiolabeled standards prepared by 5′-phosphorylationof nucleoside 3′-mono-phosphates with T4 polynucleotide kinase andγ-³²P-ATP.

FIG. 5: Synthetic 21 and 22 nt RNAs Mediate Target RNA Cleavage.

(A) Graphic representation of control 52 bp dsRNA and synthetic 21 and22 nt dsRNAs. The sense strand of 21 and 22 nt short interfering RNAs(siRNAs) is shown blue, the antisense strand in red. The sequences ofthe siRNAs were derived from the cloned fragments of 52 and 111 bpdsRNAs (FIG. 4A), except for the 22 nt antisense strand of duplex 5. ThesiRNAs in duplex 6 and 7 were unique to the 111 bp dsRNA processingreaction. The two 3′ overhanging nucleotides indicated in green arepresent in the sequence of the synthetic antisense strand of duplexes 1and 3. Both strands of the control 52 bp dsRNA were prepared by in vitrotranscription and a fraction of transcripts may contain untemplated 3′nucleotide addition. The target RNA cleavage sites-directed by the siRNAduplexes are indicated as orange circles (see legend to FIG. 4A) andwere determined as shown in FIG. 5B. (B) Position of the cleavage siteson sense and antisense target RNAs. The target RNA sequences are asdescribed in FIG. 3B. Control 52 bp dsRNA (10 nM) or 21 and 22 nt RNAduplexes 1-7 (100 nM) were incubated with target RNA for 2.5 h at 25° C.in Drosophila lysate. The stable 5′ cleavage products were resolved onthe gel. The cleavage sites are indicated in FIG. 5A. The regiontargeted by the 52 bp dsRNA or the sense (s) or antisense (as) strandsare indicated by the black bars to the side of the gel. The cleavagesites are all located within the region of identity of the dsRNAs. Forprecise determination of the cleavage sites of the antisense strand, alower percentage gel was used.

FIG. 6: Long 3′ Overhangs on Short dsRNAs Inhibit RNAi.

(A) Graphic representation of 52 bp dsRNA constructs. The 3′ extensionsof sense and antisense strand are indicated in blue and red,respectively. The observed cleavage sites on the target RNAs arerepresented as orange circles-analogous to FIG. 4A and were determinedas shown in FIG. 6B. (B) Position of the cleavage sites on sense andantisense target RNAs. The target RNA sequences are as described in FIG.3B. DsRNA (10 nM) was incubated with target RNA for 2.5h at 25° C. inDrosophila lysate. The stable 5′ cleavage products were resolved on thegel. The major cleavage sites are indicated with a horizontal arrow andalso represented in FIG. 6A. The region targeted by the 52 bp dsRNA isrepresented as black bar at both sides of. the gel.

FIG. 7: Proposed Model for RNAi.

RNAi is predicted to begin with processing of dsRNA (sense strand inblack, antisense strand in red) to predominantly 21 and 22 nt shortinterfering RNAs (siRNAs). Short overhanging 3′ nucleotides, if presenton the dsRNA, may be beneficial for processing of short dsRNAs. ThedsRNA-processing proteins, which remain to be characterized, arerepresented as green and blue ovals, and assembled on the dsRNA inasymmetric fashion. In our model, this is illustrated by binding of ahypothetical blue protein or protein domain with the siRNA strand in 3′to 5′ direction while the hypothetical green protein or protein domainis always bound to the opposing siRNA strand. These-proteins or a subsetremain associated with the siRNA duplex and preserve its orientation asdetermined by the direction of the dsRNA processing reaction. Only thesiRNA sequence associated with the blue protein is able to guide targetRNA cleavage. The endonuclease complex is referred to as smallinterfering ribonucleoprotein complex or siRNP. It is presumed here,that the endonuclease that cleaves the dsRNA may also cleave the targetRNA, probably by temporarily displacing the passive siRNA strand notused for target recognition. The target RNA is then cleaved in thecenter of the region recognized by the sequence-complementary guidesiRNA.

FIG. 8: Reporter Constructs and siRNA Duplexes.

(a) The firefly (Pp-luc) and sea pansy (Rr-luc) luciferase reporter generegions from plasmids pGL2-Control, pGL-3-Control and pRL-TK (Promega)are illustrated. SV40 regulatory elements, the HSV thymidine kinasepromoter and two introns (lines) are indicated. The sequence of GL3luciferase is 95% identical to GL2, but RL is completely unrelated toboth. Luciferase expression from pGL2 is approx. 10-fold lower than frompGL3 in transfected mammalian cells. The region targeted by the siRNAduplexes is indicated as black bar below the coding region of theluciferase genes. (b) The sense (top) and antisense (bottom) sequencesof the siRNA duplexes targeting GL2, GL3 and RL luciferase are shown.The GL2 and GL3 siRNA duplexes differ by only 3 single nucleotidesubstitutions (boxed in gray) As unspecific control, a duplex with theinverted GL2 sequence, invGL2, was synthesized. The 2 nt 3′ overhang of2′-deoxythymidine is indicated as TT; uGL2 is similar to GL2 siRNA butcontains ribo-uridine 3′ overhangs.

FIG. 9: RNA Interference by siRNA Duplexes.

Ratios of target control luciferase were normalized to a buffer control(bu, black bars); gray bars indicate ratios of Photinus pyralis (Pp-luc)GL2 or GL3 luciferase to Renilla reniformis (Rr-luc) RL luciferase (leftaxis), white bars indicate RL to GL2 or GL3-ratios (right axis), Panelsa, c, e, g and i describe experiments performed with the combination ofpGL2-Control and pRL-TK reporter plasmids, panels b, d, f, h and j withpGL3-Control and pRL-TK reporter plasmids. The cell line used for theinterference experiment is indicated at the top of each plot. The ratiosof Pp-luc/Rr-luc for the buffer control (bu) varied between 0.5 and 10for pGL2/pRL and between 0.03 and 1 for pGL3/pRL, respectively, beforenormalization and between the various cell lines tested. The plotteddata were averaged from three independent experiments ±S.D.

FIG. 10: Effects of 21 nt siRNA, 50 bp and 500 bp dsRNAs on LuciferaseExpression in HeLa Cells.

The exact length of the long dsRNAs is indicated below the bars. Panelsa, c and e describe experiments performed with pGL2-Control and PRL-TKreporter plasmids, panels b, d and f with pGL3-Control and pRL-TKreporter plasmids. The data were averaged from two independentexperiments ±S.D. (a), (b) Absolute Pp-luc expression, plotted inarbitrary luminescence units. (c), (d) Rr-luc expression, plotted inarbitrary luminescence units. (e), (f) Ratios of normalized target tocontrol luciferase. The ratios of luciferase activity for siRNA duplexeswere normalized to a buffer control (bu, black bars); the luminescenceratios for 50 or 500 bp dsRNAs were normalized to the respective ratiosobserved for 50 and 500 bp dsRNA from humanized GFP (hG, black bars). Itshould be noted that the overall differences in sequences between the 49and 484 bp dsRNAs targeting GL2 and GL3 are not sufficient to conferspecificity between GL2 and GL3 targets (43 nt uninterrupted identity in49 bp segment, 239 nt longest uninterrupted identity in 484 bp segment).

FIG. 11: Variation of the 3′ Overhang of Duplexes of 21-nt siRNAs.

(A) Outline of the experimental strategy. The capped and polyadenylatedsense target mRNA is depicted and the relative positions of sense andantisense siRNAs are shown. Eight series of duplexes, according to theeight different antisense-strands were prepared. The siRNA sequences andthe number of overhanging nucleotides were changed in 1-nt steps. (B)Normalized relative luminescence of target luciferase (Photinus pyralis,Pp-luc) to control luciferase (Renilla reniformis, Rr-luc) in D.melanogaster embryo lysate in the presence of 5 nM blunt-ended dsRNAs.The luminescence ratios determined in the presence of dsRNA werenormalized to the ratio obtained for a buffer control (bu, black bar).Normalized ratios less than 1 indicate specific interference. (C-J)Normalized interference ratios for eight series of 21-nt siRNA duplexes.The sequences of siRNA duplexes are depicted above the bar graphs. Eachpanel shows the interference ratio for a set of duplexes formed with agiven antisense guide siRNA and 5 different sense siRNAs. The number ofoverhanging nucleotides (3′ overhang, positive numbers; 5′ overhangs,negative numbers) is indicated on the x-axis. Data points were averagedfrom at least 3 independent experiments, error bars represent standarddeviations.

FIG. 12: Variation of the Length of the Sense Strand of siRNA Duplexes.

(A) Graphic representation of the experiment. Three 21-nt antisensestrands were paired with eight sense siRNAs. The siRNAs were changed inlength at their 3′ end. The 3′ overhang of the antisense siRNA was 1-nt(B), 2-nt (C), or 3-nt (D) while the sense siRNA overhang was varied foreach series. The sequences of the siRNA duplexes and the correspondinginterference ratios are indicated.

FIG. 13: Variation of the Length of siRNA Duplexes with Preserved 2-nt3′ Overhangs.

(A) Graphic representation of the experiment. The 21-nt siRNA duplex isidentical in sequence to the one shown in FIG. 11H or 12C. The siRNAduplexes were extended to the 3′ side of the sense siRNA (B) or the 5′side of the sense siRNA. (C). The siRNA duplex sequences and therespective interference ratios are indicated.

FIG. 14: Substitution of the 2′-Hydroxyl Groups of the siRNA RiboseResidues.

The 2′-hydroxyl groups (OH) in the strands of siRNA duplexes werereplaced by 2′-deoxy (d) or 2′-O-methyl (Me). 2-nt and 4-nt 2′-deoxysubstitutions at the 3′-ends are indicated as 2-nt d and 4-nt d,respectively. Uridine residues were replaced by 2′-deoxy thymidine.

FIG. 15: Mapping of Sense and Antisense Target RNA Cleavage by 21-ntsiRNA Duplexes with 2-nt 3′ Overhangs.

(A) Graphic representation of 32P (asterisk) cap-labelled sense andanti-sense target RNAs and siRNA duplexes. The position of sense andanti-sense target RNA cleavage is indicated by triangles on top andbelow the siRNA duplexes, respectively. (B) Mapping of target RNAcleavage sites. After 2 h incubation of 10 nM target with 100 nM siRNAduplex in D. melanogaster embryo lysate, the 5′ cap-labelled substrateand the 5′ cleavage products were resolved on sequencing gels. Lengthmarkers were generated by partial RNase T1 digestion (T1) and partialalkaline hydrolysis (OH—) of the target RNAs. The bold lines to the leftof the images indicate the region covered by the siRNA strands 1 and 5of the same orientation as the target.

FIG. 16: The 5′ End of a Guide siRNA Defines the Position of Target RNACleavage.

(A, B) Graphic representation of the experimental strategy. Theantisense siRNA was the same in all siRNA duplexes, but the sense strandwas varied between 18 to 25 nt by changing the 3′ end (A) or 18 to 23 ntby changing the 5′ end (B). The position of sense and antisense targetRNA cleavage is indicated by triangles on top and below the siRNAduplexes, respectively. (C, D) Analysis of target RNA cleavage usingcap-labelled sense (top panel) or antisense (bottom panel) target RNAs.Only the cap-labelled 5′ cleavage products are shown. The sequences ofthe siRNA duplexes are indicated, and the length of the sense siRNAstrands is marked on top of the panel. The control lane marked with adash in panel (C) shows target RNA incubated in absence of siRNAs.Markers were as described in FIG. 15. The arrows in (D), bottom panel,indicate the target RNA cleavage sites that differ by 1 nt.

FIG. 17: Sequence Variation of the 3′ Overhang of siRNA Duplexes.

The 2-nt 3′ overhang (NN, in gray) was changed in sequence andcomposition as indicated (T, 2′-deoxythymidine, dG, 2′-deoxyguanosine;asterisk, wild-type siRNA duplex). Normalized interference ratios weredetermined as described in FIG. 11. The wild-type sequence is the sameas depicted in FIG. 14.

FIG. 18: Sequence Specificity of Target Recognition.

The sequences of the mismatched siRNA duplexes are shown, modifiedsequence segments or single nucleotides are underlayed in gray. Thereference duplex (ref) and the siRNA duplexes 1 to 7 contain2′-deoxythymidine 2-nt overhangs. The silencing efficiency of thethymidine-modified reference duplex was comparable to the wild-typesequence (FIG. 17). Normalized interference ratios were determined asdescribed in FIG. 11.

FIG. 19: Variation of the Length of siRNA Duplexes with Preserved 2-nt3′ Overhangs.

The siRNA duplexes were extended to the 3′ side of the sense siRNA (A)or the 5′ side of the sense siRNA (B). The siRNA duplex sequences andthe respective interference ratios are indicated. For HeLa SS6 cells,siRNA duplexes (0.84 μg) targeting GL2 luciferase were transfectedtogether with pGL2-Control and pRL-TK plasmids. For comparison, the invitro RNAi activities of siRNA duplexes tested in D. melanogaster lysateare indicated.

EXAMPLE 1 RNA Interference Mediated by Small Synthetic RNAs 1.1.Experimental Procedures 1.1.1 In Vitro RNAi

In vitro RNAi and lysate preparations were performed as describedpreviously (Tuschl et al., 1999; Zamore et al., 2000). It is critical touse freshly dissolved creatine kinase (Roche) for optimal ATPregeneration. The RNAi translation assays (FIG. 1) were performed withdsRNA concentrations of 5 nM. and an extended pre-incubation period of15 min at 25° C. prior to the addition of in vitro transcribed, cappedand polyadenylated Pp-luc and Rr-luc reporter mRNAs. The incubation wascontinued for 1 h and the relative amount of Pp-luc and Rr-luc proteinwas analyzed using the dual luciferase assay (Promega) and a Monolight3010C luminometer (PharMingen).

1.1.2° RNA Synthesis

Standard procedures were used for in vitro transcription of RNA from PCRtemplates carrying T7 or SP6 promoter sequences, see for example (Tuschlet al., 1998). Synthetic RNA was prepared using Expedite RNAphosphoramidites (Proligo). The 3′ adapter oligonucleotide wassynthesized usingdimethoxytrityl-1,4-benzenedimethanol-succinyl-aminopropyl-CPG. Theoligoribonucleotides were deprotected in 3 ml of 32% ammonia/ethanol(3/1) for 4 h at 55° C. (Expedite RNA) or 16 h at 55° C. (3′ and 5′adapter DNA/RNA chimeric oligonucleotides) and then desilylated andgel-purified as described previously (Tuschl et al., 1993). RNAtranscripts for dsRNA preparation including long 3′ overhangs weregenerated from PCR templates that contained a T7 promoter in sense andan SP6 promoter in antisense direction. The transcription template forsense and antisense target RNA was PCR-amplified withGCGTAATACGACTCACTATAGAACAATTGCTTTTACAG (underlined, T7 promoter) as 5′primer and ATTTAGGTGACACTATAGGCATAAAGAATTGAAGA (underlined, SP6promoter) as 3′ primer and the linearized Pp-luc plasmid (pGEM-lucsequence) (Tuschl et al., 1999) as template; the T7-transcribed senseRNA was 17 nt long with the Pp-luc sequence between pos. 113-273relative to the start codon and followed by 17 nt of the complement ofthe SP6 promoter sequence at the 3′ end. Transcripts for blunt-endeddsRNA formation were prepared by transcription from two different PCRproducts which only contained a single promoter sequence.

DsRNA annealing was carried out using a phenol/chloroform extraction.Equimolar concentration of sense and antisense RNA (50 nM to 10 μM,depending on the length and amount available) in 0.3 M NaOAc (pH 6) wereincubated for 30 s at 90° C. and then extracted at room temperature withan equal volume of phenol/chloroform, and followed by a chloroformextraction to remove residual phenol. The resulting dsRNA wasprecipitated by addition of 2.5-3 volumes of ethanol. The pellet wasdissolved in lysis buffer (100 mM KCl, 30 mM HEPES-KOH, pH 7.4, 2 mMMg(OAc)₂) and the quality of the dsRNA was verified by standard agarosegel electrophoreses in 1×TAE-buffer. The 52 bp-dsRNAs with the 17 nt and20 nt 3′ overhangs (FIG. 6) were annealed by incubating for 1 min at 95°C., then rapidly cooled to 70° C. and followed by slow cooling to roomtemperature over a 3 h period (50 μl annealing reaction, 1 μM strandconcentration, 300 mM NaCl, 10 mM Tris-HCl, pH 7.5). The dsRNAs werethen phenol/chloroform extracted, ethanol-precipitated and dissolved inlysis buffer.

Transcription of internally ³²P-radiolabeled RNA used for dsRNApreparation (FIGS. 2 and 4) was performed using 1 mM ATP, CTP, GTP, 0.1or 0.2 mM UTP, and 0.2-0.3 μM -³²P-UTP (3000 Ci/mmol), or therespective-ratio for radiolabeled nucleoside triphosphates other thanUTP. Labeling of the cap of the target RNAs was performed as describedpreviously. The target RNAs were gel-purified after cap-labeling.

1.1.3 Cleavage Site Mapping

Standard RNAi-reactions were performed by pre-incubating 10 nM dsRNA for15 min followed by addition of 10 nM cap-labeled target RNA. Thereaction was stopped after a further 2 h (FIG. 2A) or 2.5 h incubation(FIGS. 5B and 6B) by proteinase K treatment (Tuschl et al., 1999). Thesamples were then analyzed on 8 or 10% sequencing gels The 21 and 22 ntsynthetic RNA duplexes were used at 100 nM final concentration (FIG.5B).

1.1.4 Cloning of ˜21-t RNAs

The 21 nt RNAs were produced by incubation of radiolabeled dsRNA inDrosophila lysate in absence of target RNA (200 μl reaction, 1 hincubation, 50 nM dsP111, or 100 nM dsP52 or dsP39). The reactionmixture was subsequently treated with proteinase K (Tuschl et al., 1999)and the dsRNA-processing products were separated on a denaturing 15%polyacrylamide gel. A band, including a size range of at least 18 to 24nt, was excised, eluted into 0.3 M NaCl overnight at 4° C. and insiliconized tubes. The RNA was recovered by ethanol-precipitation anddephosphorylated (30 μl reaction, 30 min, 50° C., 10 U alkalinephosphatase, Roche). The reaction was stopped by phenol/chloroformextraction and the RNA was ethanol-precipitated. The 3′ adapteroligonucleotide (pUUUaaccgcatccttctcx: upper-case, RNA; lowercase, DNA;p, phosphate; x, 4-hydroxymethylbenzyl) was then ligated to thedephosphorylated ˜21 nt RNA (20 μl reaction, 30 min, 37° C., 5 μM 3′adapter, 50 mM Tris-HCl, pH-7.6, 10 mM MgCl₂, 0.2 mM ATP, 0.1 mg/mlacetylated BSA, 15% DMSO, 25 U T4 RNA ligase, Amersham-Pharmacia) (Panand Uhlenbeck, 1992). The ligation reaction was stopped by the additionof an equal volume of 8 M urea/50 mM EDTA stopmix and directly loaded ona 15% gel. Ligation yields were greater 50%. The ligation product wasrecovered from the gel and 5′-phosphorylated (20 μl reaction, 30 min,37° C., 2 mM ATP, 5 U T4 polynucleotide kinase, NEB). Thephosphorylation reaction was stopped by phenol/chloroform extraction andRNA was recovered by ethanol-precipitation. Next, the 5′ adapter(tactaatacgactcactAAA: uppercase, RNA; lowercase, DNA) was ligated tothe phosphorylated ligation product as described above. The new ligationproduct was gel-purified and eluted from the gel slice in the presenceof reverse transcription primer (GACTAGCTGGAATTCAAGGATGCGGTTAAA: bold,Eco RI site) used as carrier. Reverse transcription (15 μl reaction, 30min, 42° C., 150 U Superscript II reverse transcriptase, LifeTechnologies) was followed by PCR using as 5′ primerCAGCCAACGGAATTCATACGACTCACTAAA (bold, Eco RI site) and the 3′ RT primer.The PCR product was purified by phenol/chloroform extraction andethanol-precipitated. The PCR product was then digested with Eco RI(NEB) and concatamerized using T4 DNA ligase (high conc., NEB).Concatamers of a size range of 200 to 800 bp were separated on alow-melt agarose gel, recovered from the gel by a standard melting andphenol extraction procedure, and ethanol-precipitated. The unpaired endswere filled in by incubation with Taq polymerase under standardconditions for 15 min at 72° C. and the DNA product was directly ligatedinto the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen).Colonies were screened using PCR and M13-20 and M13 Reverse sequencingprimers. PCR products were directly submitted for custom sequencing(Sequence Laboratories Gröttingen GmbH, Germany). On average, four tofive 21 mer sequences were obtained per clone.

1.1.5 2D-TLC Analysis

Nuclease P1 digestion of radiolabeled, gel-purified siRNAs and 2D-TLCwas carried-out as described (Zamore et al., 2000). Nuclease T2digestion was performed in 10 μl reactions for 3 h at 50° C. in 10 mMammonium acetate (pH 4.5) using 2 μg/μl carrier tRNA and 30 Uribonuclease T2 (Life Technologies). The migration of non-radioactivestandards was determined by UV shadowing. The identity ofnucleoside-3′,5′-disphosphates was confirmed by co-migration of the T2digestion products with standards prepared by 5′-³²P-phosphorylation ofcommercial nucleoside 3′-monophosphates using γ-³²P-ATP and T4polynucleotide kinase (data not shown).

1.2 Results and Discussion

1.2.1 Length Requirements for Processing of dsRNA to 21 and 22 nt RNAFragments

Lysate prepared from D. melanogaster syncytial embryos recapitulatesRNAi in vitro providing a novel tool for biochemical analysis of themechanism of RNAi (Tuschl et al., 1999; Zamore et al., 2000). In vitroand in vivo analysis of the length requirements of dsRNA for RNAi hasrevealed that short dsRNA (<150 bp) are less effective than longerdsRNAs in degrading target mRNA (Caplen et al., 2000; Hammond et al.,2000; Ngo et al., 1998); Tuschl et al., 1999). The reasons for reductionin mRNA degrading efficiency are not understood. We therefore examinedthe precise length requirement of dsRNA for target RNA degradation underoptimized conditions in the Drosophila lysate (Zamore et al., 2000).Several series of dsRNAs were synthesized and directed against fireflyluciferase (Pp-luc) reporter RNA. The specific suppression of target RNAexpression was monitored by the dual luciferase assay (Tuschl et al.,1999) (FIGS. 1A and 1B). We detected specific inhibition of target RNAexpression for dsRNAs as short as 38 bp, but dsRNAs of 29 to 36 bp werenot effective in this process. The effect was independent of the targetposition and the degree of inhibition of Pp-luc mRNA expressioncorrelated with the length of the dsRNA, i.e. long dsRNAs were moreeffective than short dsRNAs.

It has been suggested that the 21-23 nt RNA fragments generated byprocessing of dsRNAs are the mediators of RNA interference andco-suppression (Hamilton and Baulcombe, 1999; Hammond et al., 2000;Zamore et al., 2000). We therefore analyzed the rate of 21-23 ntfragment formation for a subset of dsRNAs ranging in size between 501 to29 bp. Formation of 21-23 nt fragments in Drosophila lysate (FIG. 2) wasreadily detectable for 39 to 501 bp long dsRNAs but was significantlydelayed for the 29 bp dsRNA. This observation is consistent with a roleof 21-23 nt fragments in guiding mRNA cleavage and provides anexplanation for the lack of RNAi by 30 bp dsRNAs. The length dependenceof 21-23 mer formation is likely to reflect a biologically relevantcontrol mechanism to prevent the undesired activation of RNAi by shortintramolecular base-paired structures of regular cellular RNAs.

1.2.239 bp dsRNA Mediates Target RNA Cleavage at a Single Site

Addition of dsRNA and 5′-capped target RNA to the Drosophila lysateresults in sequence-specific degradation of the target RNA (Tuschl etal., 1999). The target mRNA is only cleaved within the region ofidentity with the dsRNA and many of the target cleavage sites wereseparated by 21-23 nt (Zamore et al., 2000). Thus, the number ofcleavage sites for a given dsRNA was expected to roughly correspond tothe length of the dsRNA divided by 21. We mapped the target cleavagesites on a sense and an antisense target RNA which was 5′ radiolabeledat the cap (Zamore et al., 2000) (FIGS. 3A and 3B). Stable 5′ cleavageproducts were separated on a sequencing gel and the position of cleavagewas determined by comparison with a partial RNase T1 and an alkalinehydrolysis ladder from the target RNA.

Consistent with the previous observation (Zamore et al., 2000), alltarget RNA cleavage sites were located within the region of identity tothe dsRNA. The sense or the antisense traget was only cleaved once by 39bp dsRNA. Each cleavage site was located 10 nt from the 5′ end of theregion covered by the dsRNA (FIG. 3B). The 52 bp dsRNA, which shares thesame 5′ end with the 39 bp dsRNA, produces the same cleavage site on thesense target, located 10 nt from the 5′ end of the region of identitywith the dsRNA, in addition to two weaker cleavage sites 23 and 24 ntdownstream of the first site. The antisense target was only cleavedonce, again 10 nt from the 5′ end of the region covered by itsrespective dsRNA. Mapping of the cleavage sites for the 38 to 49 bpdsRNAs shown in FIG. 1 showed that the first and predominant cleavagesite was always located 7 to 10 nt downstream of the region covered bythe dsRNA (data not shown). This suggests that the point of target RNAcleavage is determined by the end of the dsRNA and could imply thatprocessing to 21-23 mers starts from the ends of the duplex.

Cleavage sites on sense and antisense target for the longer 111 bp dsRNAwere much more frequent than anticipated and most of them appear inclusters separated by 20 to 23 nt (FIGS. 3A and 3B). As for the shorterdsRNAs, the first cleavage site on the sense target is 10 nt from the 5′end of the region spanned by the dsRNA, and the first cleavage site onthe antisense target is located 9 nt from the 5′ end of region coveredby the dsRNA. It is unclear what causes this disordered cleavage, butone possibility could be that longer dsRNAs may not only get processedfrom the ends but also internally, or there are some specificitydeterminants for dsRNA processing which we do not yet understand. Someirregularities to the 21-23 nt-spacing were also previously noted(Zamore et al., 2000). To better understand the molecular basis of dsRNAprocessing and target RNA recognition, we decided to analyze thesequences of the 21-23 nt fragments generated by processing of 39, 52,and 111 bp dsRNAs in the Drosophila lysate.

1.2.3 dsRNA is Processed to 21 and 22 nt RNAs by an RNase III-LikeMechanism

In order to characterize the 21-23 nt RNA fragments we examined the 5′and 3′ termini of the RNA fragments. Periodate oxidation of gel-purified21-23 nt RNAs followed by β-elimination indicated the presence of aterminal 2′ and 3′ hydroxyl groups. The 21-23 mers were also responsiveto alkaline phosphatase treatment indicating the presence of a 5′terminal phosphate group. The presence of 5′ phosphate and 3′ hydroxyltermini suggests that the dsRNA could be processed by an enzymaticactivity similar to E. coli RNase III (for reviews, see (Dunn, 1982;Nicholson, 1999; Robertson, 1990; Robertson, 1982)).

Directional cloning of 21-23 nt RNA fragments was performed by ligationof a 3′ and 5′ adapter oligonucleotide to the purified 21′-23 mers usingT4 RNA ligase. The ligation products were reverse transcribed,PCR-amplified, concatamerized, cloned, and sequenced. Over 220 shortRNAs were sequenced from dsRNA processing reactions of the 39, 52 and111bp dsRNAs (FIG. 4A). We found the following length distribution: 1%18 nt, 5% 19 nt, 12% 20 nt, 45% 21 nt, 28% 22 nt, 6% 23 nt, and 2% 24nt. Sequence analysis of the 5′ terminal nucleotide of the processedfragments indicated that oligonucleotides with a 5′ guanosine wereunderrepresented. This bias was most likely introduced by T4 RNA ligasewhich discriminates against 5′ phosphorylated guanosine as donoroligonucleotide; no significant sequence bias was seen at the 3′ end.Many of the ˜21 nt fragments derived from the 3′ ends of the sense orantisense strand of the duplexes include 3′ nucleotides that are derivedfrom untemplated addition of nucleotides during RNA synthesis using T7RNA polymerase. Interestingly, a significant number of endogenousDrosophila ˜21 nt RNAs were also cloned, some of them from LTR andnon-LTR retrotransposons (data not shown). This is consistent with apossible role for RNAi in transposon silencing.

The ˜21 nt RNAs appear in clustered groups (FIG. 4A) which cover theentire dsRNA sequences. Apparently, the processing reaction cuts thedsRNA by leaving staggered 3′ ends, another characteristic of RNase IIIcleavage. For the 39 bp dsRNA, two clusters of ˜21 nt RNAs we re foundfrom each dsRNA-constituting strand including overhanging 3′ ends, yetonly one cleavage site was detected on the sense and antisense target(FIGS. 3A and 3B). If the ˜21 nt fragments were present assingle-stranded guide RNAs in a complex that mediates mRNA degradation,it could be assumed that at least two target cleavage sites exist, butthis was not the case. This suggests that the ˜21 nt RNAs may be presentin double-stranded form in the endonuclease complex but that only one ofthe strands can be used for target RNA recognition and cleavage. The useof only one of the ˜21 nt strands for target cleavage may simply bedetermined by the orientation in which the ˜21 nt duplex is bound to thenuclease complex. This orientation is defined by the direction in whichthe original dsRNA was processed.

The ˜21mer clusters for the 52 bp and 111 bp dsRNA are less well definedwhen compared to the 39 bp dsRNA. The clusters are spread over regionsof 25 to 30 nt most likely representing several distinct subpopulationsof ˜21 nt duplexes and therefore guiding target cleavage at severalnearby sites. These cleavage regions are still predominantly separatedby 20 to 23 nt intervals. The rules determining how regular dsRNA can beprocessed to ˜21 nt fragments are not yet understood, but it waspreviously observed that the approx. 21-23 nt spacing of cleavage sitescould be altered by a run of uridines (Zamore et al., 2000). Thespecificity of 5 dsRNA cleavage by E. coli RNase III appears to bemainly controlled by antideterminants, i.e. excluding some specificbase-pairs at given positions relative to the cleavage site (Zhang andNicholson, 1997).

To test whether sugar-, base- or cap-modification were present inprocessed ˜21 nt RNA fragments, we incubated radiolabeled 505 bp Pp lucdsRNA in lysate for 1 h, isolated the ˜21 nt products, and digested itwith P1 or T2 nuclease to mononucleotides. The nucleotide mixture wasthen analyzed by 2D thin-layer chromatography (FIG. 4B). None of thefour natural ribonucleotides were modified as indicated by P1 or T2digestion. We have previously analyzed adenosine to inosine conversionin the ˜21 nt fragments (after a 2 h incubation) and detected a smallextent (<0.7%) deamination (Zamore et al., 2000); shorter incubation inlysate (1 h) reduced this inosine fraction to barely detectable levels.RNase T2, which cleaves 3′ of the phosphodiester linkage, producednucleoside 3′-phosphate and nucleoside 3′,5′-diphosphate, therebyindicating the presence of a 5′-terminal monophosphate. All fournucleoside 3′,5′-diphosphates were detected and suggest that theinternucleotidic linkage was cleaved with little or nosequence-specificity. In summary, the ˜21 nt fragments are unmodifiedand were generated from dsRNA such that 5′-monophosphates and3′-hydroxyls were present at the 5′-end.

1.2.4 Synthetic 21 and 22 nt RNAs Mediate Target RNA Cleavage

Analysis of the products of dsRNA processing indicated that the ˜21 ntfragments are generated by a reaction with all the characteristics of anRNase III cleavage reaction (Dunn, 1982; Nicholson, 1999; Robertson,1990; Robertson, 1982). RNase III makes two staggered cuts in bothstrands of the dsRNA, leaving a 3′ overhang of about 2 nt. We chemicallysynthesized 21 and 22 nt RNAs, identical in sequence to some of thecloned ˜21 nt fragments, and tested them for their ability to mediatetarget RNA degradation (FIGS. 5A and 5B). The 21 and 22 nt RNA duplexeswere incubated at 100 nM concentrations in the lysate, a 10-fold higherconcentrations than the 52 bp-control dsRNA. Under these conditions,target RNA cleavage is readily detectable. Reducing the concentration of21 and 22 nt duplexes from 100 to 10 nM does still cause target RNAcleavage. Increasing the duplex concentration from 100 nM to 1000 nMhowever does not further increase target cleavage, probably due to alimiting protein factor within the lysate.

In contrast to 29 or 30 bp dsRNAs that did not mediate RNAi, the 21 and22 nt dsRNAs with overhanging 3′ ends of 2 to 4 nt mediated efficientdegradation of target RNA (duplexes 1, 3, 4, 6, FIGS. 5A and 5B).Blunt-ended 21 or 22 nt dsRNAs (duplexes 2, 5, and 7, FIGS. 5A and 5B)were reduced in their ability to degrade the target and indicate thatoverhanging 3′ ends are critical for reconstitution of theRNA-protein-nuclease complex. The single-stranded overhangs may berequired for high affinity-binding of the ˜21 nt duplex to the proteincomponents. A 5′ terminal phosphate, although present after dsRNAprocessing, was not required to mediate target RNA cleavage and wasabsent from the short synthetic RNAs.

The synthetic 21 and 22 nt duplexes guided cleavage of sense as well asantisense targets within the region covered by the short duplex. This isan important result considering that a 39 bp dsRNA which forms two pairsof clusters of ˜21 nt fragments (FIG. 2), cleaved sense or antisensetarget only once and not twice. We interpret this result by suggestingthat only one of two strands present in the ˜21 nt duplex is able toguide target RNA cleavage and that the orientation of the ˜21 nt duplexin the nuclease complex is determined by the initial direction of dsRNAprocessing.

The presentation of an already perfectly processed ˜21 nt duplex to thein vitro system however does allow formation of the activesequence-specific nuclease complex with two possible orientations of thesymmetric RNA duplex. This results in cleavage of sense as well asantisense target within the region of identity with the 21 nt RNAduplex.

The target cleavage site is located 11 or 12 nt downstream of the firstnucleotide that is complementary to the 21 or 22 nt guide sequence, i.e.the cleavage site is near center of the region covered by the 21 or 22nt RNAs (FIGS. 4A and 4B). Displacing the sense strand of a 22 nt duplexby two nucleotides (compare duplexes 1 and 3 in FIG. 5A) displaced thecleavage site of only the antisense target by two nucleotides.Displacing both sense and antisense strand by two nucleotides shiftedboth cleavage sites by two nucleotides (compare duplexes 1 and 4). Wepredict that it will be possible to design a pair of 21 or 22 nt RNAs tocleave a target RNA at almost any given: position.

The specificity of target RNA cleavage guided by 21 and 22 nt RNAsappears exquisite as no aberrant cleavage sites are detected (FIG. 5B).It should however be noted, that the nucleotides present in the 3′overhang. of the 21 and 22 nt RNA duplex may contribute less tosubstrate recognition than the nucleotides near the cleavage site. Thisis based on the observation that the 3′ most nucleotide in the 3′overhang of the active duplexes 1 or 3 (FIG. 5A) is not complementary tothe target. A detailed analysis of the specificity of RNAi can now bereadily undertaken using synthetic 21 and 22 nt RNAs.

Based on the evidence that synthetic 21 and 22 nt RNAs with overhanging3′ ends mediate RNA interference, we propose to name the ˜21 nt RNAs“short interfering RNAs” or siRNAs and the respective RNA-proteincomplex a “small interfering ribonucleoprotein particle” or siRNP.

1.2.5 3′ Overhangs of 20 Nt on Short dsRNAs Inhibit RNAi

We have shown that short blunt-ended dsRNAs appear to be processed fromthe ends of the dsRNA. During our study of the length dependence ofdsRNA in RNAi, we have also analyzed dsRNAs with 17 to 20 nt-overhanging3′ ends and found to our surprise that they were less potent thanblunt-ended dsRNAs. The inhibitory effect of long 3′ ends wasparticularly pronounced for dsRNAs up to 100 bp but was less dramaticfor longer dsRNAs. The effect was not due to imperfect dsRNA formationbased on native gel analysis (data not shown). We tested if theinhibitory effect of long overhanging 3′ ends could be used as a tool todirect dsRNA processing to only one of the two ends of a short RNAduplex.

We synthesized four combinations of the 52 bp model dsRNA, blunt-ended,3′ extension on only the sense strand, 3′-extension on only theantisense strand, and double 3′ extension on both strands, and mappedthe target RNA cleavage sites after incubation in lysate (FIGS. 6A and6B). The first and predominant cleavage site of the sense target waslost when the 3′ end of the antisense strand of the duplex was extended,and vice versa, the strong cleavage site of the antisense target waslost when the 3′ end of sense strand of the duplex was extended. 3′Extensions on both strands rendered the 52 bp dsRNA virtually inactive.One explanation for the dsRNA inactivation by ˜20 nt 3′ extensions couldbe the association of single-stranded RNA-binding proteins which couldinterfere with the association of one of the dsRNA-processing factors atthis end. This result is also consistent with our model where only oneof the strands of the siRNA duplex in the assembled siRNP is able toguide target RNA cleavage. The orientation of the strand that guides RNAcleavage is defined by the direction of the dsRNA processing reaction.It is likely that the presence of 3′ staggered ends may facilitate theassembly of the processing complex. A block at the 3′ end of the sensestrand will only permit dsRNA processing from the opposing 3′ end of theantisense strand. This in turn generates siRNP complexes in which onlythe antisense strand of the siRNA duplex is able to guide sense targetRNA cleavage. The same is true for the reciprocal situation.

The less pronounced inhibitory effect of long 3′ extensions in the caseof longer dsRNAs (>500 bp, data not shown) suggests to us that longdsRNAs may also contain internal dsRNA-processing signals or may getprocessed cooperatively due to the association of multiple cleavagefactors.

1.2.6 A Model for dsRNA-Directed mRNA. Cleavage

The new biochemical data update the model for how dsRNA targets mRNA fordestruction (FIG. 7). Double-stranded RNA is first processed to shortRNA duplexes of predominantly 21 and 22 nt in length and with staggered3′ ends similar to an RNase III-like reaction (Dunn, 1982; Nicholson,1999; Robertson, 1982). Based on the 21-23 nt length of the processedRNA fragments it has already been speculated that an RNase III-likeactivity may be involved in RNAi (Bass, 2000). This hypothesis isfurther supported by the presence of 5′ phosphates and 3′ hydroxyls atthe termini of the siRNAs as observed in RNase III reaction products(Dunn, 1982; Nicholson, 1999). Bacterial RNase III and the eukaryotichomologs Rnt1p in S. cerevisiae and Pac1p in S. pombe have been shown tofunction in processing of ribosomal RNA as well as snRNA and snoRNAs(see for example Chanfreau et al., 2000).

Little is known about the biochemistry of RNase III homologs fromplants, animals or human. Two families of RNase ill enzymes have beenidentified predominantly by database-guided sequence analysis or cloningof cDNAs. The first RNase. III family is represented by the 1327 aminoacid long D. melanogaster protein drosha (Acc. AF116572). The C-terminusis composed of two RNase III and one dsRNA-binding domain and theN-terminus is of unknown function. Close-homologs are also found in C.elegans (Acc. AF160248) and human (Acc. AF189011) (Filippov et al.,2000; Wu et al., 2000). The drosha-like human RNase III was recentlycloned and characterized (Wu et al., 2000). The gene is ubiquitouslyexpressed in human tissues and cell lines, and the protein is localizedin the nucleus and the nucleolus of the cell. Based on results inferredfrom antisense inhibition studies, a role of this protein for rRNAprocessing was suggested. The second class is represented by the C.elegans gene K12H4.8 (Acc. S44849) coding for a 1822 amino acid longprotein. This protein has an N-terminal RNA helicase motif which isfollowed by 2 RNase III catalytic domains and a dsRNA-binding motif,similar to the drosha RNase III family. There are close homologs in S.pombe (Acc. Q09884), A. thaliana (Acc. AF187317), D. melanogaster (Acc.AE003740), and human (Acc. AB028449) (Filippov et al., 2000; Jacobsen etal., 1999; Matsuda et al., 2000). Possibly the K12H4.8 RNaseIII/helicase is the likely candidate to be involved in RNAi.

Genetic screens in C. elegans identified rde-1 and rde-4-as essentialfor activation of RNAi without an effect on transposon mobilization- orco-suppression (Dernburg et al., 2000; Grishok et al., 2000; Ketting andPlasterk, 2000; Tabara et al., 1999). This led to the hypothesis thatthese genes are important for dsRNA processing but are not involved inmRNA target degradation. The function of both genes is as yet unknown,the rde-1 gene product is a member of a family of proteins similar tothe rabbit protein eIF2C (Tabara et al., 1999), and the sequence ofrde-4 has not yet been described. Future biochemical characterization ofthese proteins should reveal their molecular function.

Processing to the siRNA duplexes appears to start from the ends of bothblunt-ended dsRNAs or dsRNAs with short (1-5 nt) 3′ overhangs, andproceeds in approximately 21-23 nt steps. Long (˜20 nt) 3′ staggeredends on short dsRNAs suppress RNAi, possibly through interaction withsingle-stranded RNA-binding proteins. The suppression of RNAi bysingle-stranded regions flanking short dsRNA and the lack of siRNAformation from short 30 bp dsRNAs may explain why structured regionsfrequently encountered in mRNAs do not lead to activation of RNAi.

Without wishing to be bound by theory, we presume that thedsRNA-processing proteins or a subset of these remain associated withthe siRNA duplex after the processing reaction. The orientation of thesiRNA duplex relative to these proteins determines which of the twocomplementary strands functions in guiding target RNA degradation.Chemically synthesized siRNA duplexes guide cleavage of sense as well asantisense target RNA as they are able- to associate with the proteincomponents in either of the two possible orientation.

The remarkable finding that synthetic 21 and 0.22 nt siRNA duplexes canbe used for efficient mRNA degradation provides new tools forsequence-specific regulation of gene expression in functional genomicsas well as biomedical studies. The siRNAs may be effective in mammaliansystems where long dsRNAs cannot be used due to the activation of thePKR response (Clemens, 1997). As such, the siRNA duplexes represent anew alternative to antisense or ribozyme therapeutics.

EXAMPLE 2 RNA Interference in Human Tissue Cultures 2.1 Methods 2.1.1RNA Preparation

21 nt RNAs were chemically synthesized using Expedite RNAphosphoramidites and thymidine phosphoramidite (Proligo, Germany).Synthetic oligonucleotides were deprotected and gel-purified (Example1), followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA)purification (Tuschl, 1993). The siRNA sequences targeting GL2 (Acc.X65324) and GL3 luciferase (Acc. U47296) corresponded to the codingregions 153-173 relative to the first nucleotide of the start codon,siRNAs targeting RL (Acc. AF025846) corresponded to region 119-129 afterthe start codon. Longer RNAs were transcribed with T7 RNA polymerasefrom PCR products, followed by gel and Sep-Pak purification. The 49 and484 bp GL2 or GL3 dsRNAs corresponded to position 113-161 and 113-596,respectively, relative to the start of translation; the 50 and 501 bp RLdsRNAs corresponded to position 118-167 and 118-618, respectively. PCRtemplates for dsRNA synthesis targeting humanized GFP (hG) wereamplified from pAD3 (Kehlenbach, 1998), whereby 50 and 501 bp hG dsRNAcorresponded to position 118-167 and 118-618, respectively, to the startcodon.

For annealing of siRNAs, 20 μM single strands were incubated inannealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2mM magnesium acetate) for 1 min at 90° C. followed by 1 h at 37° C. The37° C. incubation step was extended overnight for the 50 and 500 bpdsRNAs and these annealing reactions were performed at 8.4 μM and 0.84μM strand concentrations, respectively.

2.1.2 Cell Culture

S2 cells were propagated in Schneider's Drosophila medium (LifeTechnologies) supplemented with 10% FBS, 100 units/ml penicillin and 100μg/ml streptomycin at 25° C. 293, NIH/3T3, HeLa S3, COS-7 cells weregrown at 37° C. in Dulbecco's modified Eagle's medium supplemented with10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. Cells wereregularly passaged to maintain exponential growth. 24 h beforetransfection at approx. 80% confluency, mammalian cells were trypsinizedand diluted 1:5 with fresh medium without antibiotics (1-3×10⁵ cells/ml)and transferred to 24-well plates (500 μl/well). S2 cells were nottrypsinized before splitting. Transfection was carried out withLipofectamine 2000 reagent (Life Technologies) as described by themanufacturer for adherent cell lines. Per well, 1.0 μg pGL2-Control(Promega) or pGL3-Control (Promega), 0.1 μg pRL-TK (Promega) and 0.28 μgsiRNA duplex or dsRNA, formulated into liposomes, were applied; thefinal volume was 600 μl per well. Cells were incubated 20 h aftertransfection and appeared healthy thereafter. Luciferase expression wassubsequently monitored with the Dual luciferase assay (Promega).Transfection efficiencies were determined by fluorescence microscopy formammalian cell lines after co-transfection of 1.1 μg hGFP-encoding pAD3and 0.28 μg invGL2 in GL2siRNA and were 70-90%. Reporter plasmids wereamplified in XL-1 Blue (Stratagene) and purified using the QiagenEndoFree Maxi Plasmid Kit.

2.2 Results and Discussion

To test whether siRNAs are also capable of mediating RNAi in tissueculture, we synthesized 21 nt sIRNA duplexes with symmetric 2 nt 3′over-hangs directed against reporter genes coding for sea pansy (Renillareniformis) and two sequence variants of firefly-(Photinus pyralis, GL2and GL3) luciferases (FIG. 8 a, b). The siRNA duplexes wereco-transfected with the reporter plasmid combinations pGL2/pRL orpGL3/pRL into D. melanogaster Schneider S2 cells or mammalian cellsusing cationic liposomes. Luciferase activities were determined 20 hafter transfection. In all cell lines tested, we observed specificreduction of the expression of the reporter genes in the presence ofcognate siRNA duplexes (FIG. 9 a-j). Remarkably, the absolute luciferaseexpression levels were unaffected by non-cognate siRNAs, indicating theabsence of harmful side effects by 21 nt RNA duplexes (e.g. FIG. 10 a-dfor HeLa cells). In D. melanogaster S2 cells (FIG. 9 a, b), the specificinhibition of luciferases was complete. In mammalian cells, where thereporter genes were 50- to 100-fold stronger expressed, the specificsuppression was less complete (FIG. 9 c-j). GL2 expression was reduced3- to 12-fold, GL3 expression 9- to 25-fold and RL expression 1- to3-fold, in response to the cognate siRNAs. For 293 cells, targeting ofRL luciferase by RL siRNAs was ineffective, although GL2 and GL3 targetsresponded specifically (FIG. 9 i, j). The lack of reduction of RLexpression in 293 cells may be due to its 5- to 20-fold higherexpression compared to any other mammalian cell line tested- and/or tolimited accessibility of the target sequence due to RNA secondarystructure or associated proteins. Nevertheless, specific targeting ofGL2 and GL3 luciferase by the cognate siRNA duplexes indicated that RNAiis also functioning in 293 cells.

The 2 nt 3′ overhang in all siRNA-duplexes, except for uGL2, wascomposed of (2′-deoxy) thymidine. Substitution of uridine by thymidinein the 3′ overhang was well tolerated in the D. melanogaster in vitrosystem and the sequence of the overhang was uncritical for targetrecognition. The thymidine overhang was chosen, because it is supposedto enhance nuclease resistance of siRNAs in the tissue culture mediumand within transfected cells. Indeed, the thymidine-modified GL2 siRNAwas slightly more potent than the unmodified uGL2 siRNA in all celllines tested (FIG. 9 a, c, e, g, i). It is conceivable that furthermodifications of the 3′overhanging nucleotides may provide additional,benefits to the delivery and stability of siRNA duplexes.

In co-transfection experiments, 25 nM siRNA duplexes with respect to thefinal volume of tissue culture medium were used (FIG. 9, 10). Increasingthe siRNA concentration to 100 nM did not enhance the specific silencingeffects, but started to affect transfection efficiencies due tocompetition for liposome encapsulation between plasmid DNA and siRNA(data not shown). Decreasing the siRNA concentration to 1.5 nM did notreduce the specific silencing effect (data not shown), even though thesiRNAs were now only 2- to 20-fold more concentrated than the DNAplasmids. This indicates that siRNAs are extraordinarily powerfulreagents for mediating gene silencing and that siRNAs are effective atconcentrations that are several orders of magnitude below theconcentrations applied in conventional antisense or ribozyme genetargeting experiments.

In order to monitor the effect of longer dsRNAs on mammalian cells, 50and 500 bp dsRNAs cognate to the reporter genes were prepared. Asnon-specific control, dsRNAs from humanized GFP (hG) (Kehlenbach, 1998)was used. When dsRNAs were co-transfected, in identical amounts (notconcentrations) to the siRNA duplexes, the reporter gene expression wasstrongly and unspecifically reduced. This effect is illustrated for HeLacells as a representative example (FIG. 10 a-d). The absolute luciferaseactivities were decreased unspecifically 10- to 20-fold by 50 bp dsRNAand 20- to 200-fold by 500 bp dsRNA co-transfection, respectively.Similar unspecific effects were observed for COS-7 and NIH/3T3 cells.For 293 cells, a 10- to 20-fold unspecific reduction was observed onlyfor 500 bp dsRNAs. Unspecific reduction in reporter gene expression bydsRNA>30 bp was expected as part of the interferon response.

Surprisingly, despite the strong unspecific decrease in reporter geneexpression, we reproducibly detected additional sequence-specific, dsRNAmediated silencing. The specific silencing effects, however, were onlyapparent when the relative reporter gene activities were normalized tothe hG dsRNA controls (FIG. 10 e, f). A 2- to 10-fold specific reductionin response to cognate dsRNA was observed, also in the other threemammalian cell lines tested (data not shown). Specific silencing effectswith dsRNAs (356-1662 bp) were previously reported in CHO—K1 cells, butthe amounts of dsRNA required to detect a 2- to 4-fold specificreduction were about 20-fold higher than in our experiments (Ui-Tei,2000). Also CHO—K1 cells appear to be deficient in the interferonresponse. In another report, 293, NIH/3T3 and BHK-21 cells were testedfor RNAi using luciferase/lacZ reporter combinations and 829 bp specificlacZ or 717 bp unspecific GFP dsRNA (Caplen, 2000). The failure ofdetecting RNAi in this case may be due to the less sensitiveluciferase/lacZ reporter assay and the length differences of target andcontrol dsRNA. Taken together, our results indicate that RNAi is activein mammalian cells, but that the silencing effect is difficult todetect, if the interferon system is activated by dsRNA>30 bp.

In summary, we have demonstrated for the first time siRNA-mediated genesilencing in mammalian cells. The use of short siRNAs holds greatpromise for inactivation of gene function in human tissue culture andthe development of gene-specific therapeutics.

EXAMPLE 3 Specific Inhibition of Gene Expression by RNA Interference 3.1Materials and Methods 3.1.1 RNA Preparation and RNAi Assay

Chemical RNA synthesis, annealing, and luciferase-based RNAi assays wereperformed as described in Examples 1 or 2 or in previous publications(Tuschl et al., 1999; Zamore et al., 2000). All siRNA duplexes weredirected against firefly luciferase, and the luciferase mRNA sequencewas derived from pGEM-luc (GenBank acc. X65316) as described (Tuschl etal., 1999). The siRNA duplexes were incubated in D. melanogasterRNAi/translation reaction for 15 min prior to addition of mRNAs.Translation-based RNAi assays were performed at least in triplicates.

For mapping of sense target RNA cleavage, a 177-nt transcript wasgenerated, corresponding to the firefly luciferase sequence betweenpositions 113-273 relative to the start codon, followed by the 17-ntcomplement of the SP6 promoter sequence. For mapping of antisense targetRNA cleavage, a 166-nt transcript was produced from a template, whichwas amplified from plasmid sequence by PCR using 5′ primerTAATACGACTCACTATAGAGCCCATATCGTTTCATA (T7 promoter underlined) and 3′primer AGAGGATGGAACCGCTGG. The target sequence corresponds to thecomplement of the firefly luciferase sequence between positions 50-215relative to the start codon. Guanylyl transferase labelling wasperformed as previously described (Zamore et al., 2000). For mapping oftarget RNA cleavage, 100 nM siRNA duplex was incubated with 5 to 10 nMtarget RNA in D. melanogaster embryo lysate under standard conditions(Zamore et al., 2000) for 2 h at 25° C. The reaction was stopped by theaddition of 8 volumes of proteinase K buffer (200 mM Tris-HCl pH 7.5, 25mM EDTA, 300 mM NaCl, 2% w/v sodium dodecyl sulfate). Proteinase K (E.M. Merck, dissolved in water) was added to a final concentration of 0.6mg/ml. The reactions were then incubated for 15 min at 65° C., extractedwith phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 3volumes of ethanol. Samples were located on 6% sequencing gels. Lengthstandards were generated by partial RNase T1 digestion and partial basehydrolysis of the cap-labelled sense or antisense target RNAs.

3.2 Results

3.2.1 Variation of the 3′ overhang in duplexes of 21-nt siRNAs

As described above, 2 or 3 unpaired nucleotides at the 3′ end of siRNAduplexes were more efficient in target RNA degradation than therespective blunt-ended duplexes. To perform a more comprehensiveanalysis of the function of the terminal nucleotides, we synthesizedfive 21-nt sense siRNAs, each displayed by one nucleotide relative tothe target RNA, and eight 21-nt antisense siRNAs, each displaced by onenucleotide relative to the target (FIG. 11A). By combining sense andantisense siRNAs, eight series of siRNA duplexes with syntheticoverhanging ends were generated covering a range of 7-nt 3′ overhang to4-nt 5′ overhang. The interference of siRNA duplexes was measured usingthe dual luciferase assay system (Tuschl et al., 1999; Zamore et al.,2000). siRNA duplexes were directed against firefly luciferase mRNA, andsea pansy luciferase mRNA was used as internal control. The luminescenceratio of target to control luciferase activity was determined in thepresence of siRNA duplex and was normalized to the ratio observed in theabsence of dsRNA. For comparison, the interference ratios of long dsRNAs(39 to 504 pb) are shown in FIG. 11B. The interference ratios weredetermined at concentrations of 5 nM for long dsRNAs (FIG. 11A) and at100 nM for siRNA duplexes (FIG. 11C-J). The 100 nM concentrations ofsiRNAs was chosen, because complete processing of 5 nM 504 bp dsRNAwould result in 120 nM total siRNA duplexes.

The ability of 21-nt siRNA duplexes to mediate RNAi is dependent on thenumber of overhanging nucleotides or base pairs formed. Duplexes withfour to six 3′ overhanging nucleotides were unable to mediate RNAi (FIG.11C-F), as were duplexes with two or more 5′ overhanging nucleotides(FIG. 11G-J). The duplexes with 2-nt 3′ overhangs were most efficient inmediating RNA interference, though the efficiency of silencing was alsosequence-dependent, and up to 12-fold differences were observed fordifferent siRNA duplexes with 2-nt 3′ overhangs (compare FIG. 11D-H).Duplexes with blunted ends, 1-nt 5′ overhang or 1- to 3-nt 3′ overhangswere sometimes functional. The small silencing effect observed for thesiRNA duplex with 7-nt 3′ overhang (FIG. 11C) may be due to an antisenseeffect of the long 3′ overhang rather than due to RNAi. Comparison ofthe efficiency of RNAi between long dsRNAs (FIG. 11B) and the mosteffective 21-nt siRNA duplexes (FIG. 11E, G, H) indicates that a singlesiRNA duplex at 100 nM concentration can be as effective as 5 nM 504 bpdsRNA.

3-2.2 Length Variation of the Sense siRNA Paired to an Invariant 21-ntAntisense siRNA

In order to investigate the effect of length of siRNA on RNAi, weprepared 3 series of siRNA duplexes, combining three 21-nt antisensestrands, with eight, 18- to 25-nt sense strands. The 3′ overhang of theantisense siRNA was fixed to 1, 2, or 3 nt in each siRNA duplex series,while the sense siRNA was varied at its 3′ end (FIG. 12A). Independentof the length of the sense siRNA, we found that duplexes with 2-nt 3′overhang of anti-sense siRNA (FIG. 12C) were more active than those with1- or 3-nt 3′ overhang (FIG. 12B, D). In the first series, with 1′-nt 3′overhang of antisense siRNA, duplexes with a 21- and 22-nt sense siRNAs,carrying a 1- and 2-nt 3′overhang of sense siRNA, respectively, weremost active. Duplexes with 19- to 25-nt sense siRNAs were also able tomediate RNA, but to a lesser extent. Similarly, in the second series,with 2-nt overhang of antisense siRNA, the 21-nt siRNA duplex with 2-nt3′ overhang was most active, and any other combination with the 18- to25-nt sense siRNAs was active to a significant degree. In the lastseries, with 3-nt anti-sense siRNA 3′ overhang, only the duplex with a20-nt sense siRNA and the 2-nt sense 3′ overhang was able to reducetarget RNA expression. Together, these results indicate that the lengthof the siRNA as well as the length of the 3′ overhang are important, andthat duplexes of 21-nt siRNAs with 2-nt 3′ overhang are optimal forRNAi.

3.2.3 Length Variation of siRNA Duplexes with a Constant 2-Nt 3′Overhang

We then examined the effect of simultaneously changing the length ofboth siRNA strands by maintaining symmetric 2-nt 3′ overhangs (FIG.13A). Two series of siRNA duplexes were prepared including the 21-ntsiRNA duplex of FIG. 11H as reference. The length of the duplexes wasvaried between 20 to 25 bp by extending the base-paired segment at the3′ end of the sense siRNA (FIG. 13B) or at the 3′ end of the antisensesiRNA (FIG. 13C). Duplexes of 20 to 23 bp caused specific repression oftarget luciferase activity, but the 21-nt siRNA duplex was at least8-fold more efficient than any of the other duplexes. 24- and 25-ntsiRNA duplexes did not result in any detectable interference.Sequence-specific effects were minor as variations on both ends of theduplex produced similar effects.

3.2.4 2′-Deoxy and 2′-O-methyl-modified siRNA Duplexes

To assess the importance of the siRNA ribose residues for RNAi, duplexeswith 21-nt siRNAs and 2-nt. 3′ overhangs with 2′-deoxy- or2—O-methyl-modified strands were examined (FIG. 14). Substitution of the2-nt 3′ overhangs by 2′-deoxy nucleotides had no effect, and even thereplacement of two additional ribonucleotides adjacent to the overhangsin the paired region, produced significantly active siRNAs. Thus, 8 outof 42 nt of a siRNA duplex were replaced by DNA residues without loss ofactivity. Complete substitution of one or both siRNA strands by 2′-deoxyresidues, however, abolished RNAi, as did substitution by 2′-O-methylresidues.

3.2.5 Definition of Target RNA Cleavage Sites

Target RNA cleavage positions-were previously determined for 22-nt siRNAduplexes and for a 21-nt/22-nt duplex. It was found that the position ofthe target RNA cleavage was located in the centre of the region coveredby the siRNA duplex, 11 or 12 nt downstream of the first nucleotide thatwas complementary to the 21- or 22-nt siRNA guide sequence. Fivedistinct 21-nt siRNA duplexes with 2-nt 3′ overhang (FIG. 15A) wereincubated with 5′ cap-labelled sense or antisense target RNA in D.melanogaster lysate (Tuschl et al., 1999; Zamore et al., 2000). The 5′cleavage products were resolved on sequencing gels (FIG. 15B). Theamount of sense target RNA cleaved correlates with the efficiency ofsiRNA duplexes determined in the translation-based assay, and siRNAduplexes 1, 2 and 4 (FIGS. 15B and 11H, G, E) cleave target RNA fasterthan duplexes-3 and 5 (FIGS. 15B and 11F, D). Notably, the sum ofradioactivity of the 5′ cleavage product and the input target RNA werenot constant over time, and the 5′ cleavage products did not accumulate.Presumably, the cleavage products, once released from thesiRNA-endonuclease complex, are rapidly degraded due to the lack ofeither of the poly(A) tail of the 5′-cap.

The cleavage sites for both, sense and antisense target RNAs werelocated in the middle of the region spanned by the siRNA duplexes. Thecleavage sites for each target produced by the 5 different duplexesvaried by 1-nt according to the 1-nt displacement of the duplexes alongthe target sequences. The targets were cleaved precisely 11 ntdownstream of the target position complementary to the 3′-mostnucleotide of the sequence-complementary guide siRNA (FIG. 1-5A, B).

In order to determine, whether the 5′ or the 3′ end of the guide siRNAsets the ruler for target RNA cleavage, we devised the experimentalstrategy outlined in FIGS. 16A and B. A 21-nt antisense siRNA, which waskept invariant for this study, was paired with sense siRNAs that weremodified at either of their 5′ or 3′ ends. The position of sense andantisense target RNA cleavage was determined as described above. Changesin the 3′ end of the sense siRNA, monitored for 1-nt 5′ overhang to 6-nt3′ overhang, did neither effect the position of sense nor antisensetarget RNA cleavage (FIG. 16C). Changes in the 5′ end of the sense siRNAdid no affect the sense target RNA cleavage (FIG. 16D, top panel), whichwas expected because the antisense siRNA was unchanged. However, theantisense target RNA cleavage was affected and strongly dependent on the5′ end of the sense siRNA (FIG. 16D, bottom panel). The antisense targetwas only cleaved, when the sense siRNA was 20 or 21 nt in size, and theposition of cleavage different by 1-nt, suggesting that the 5′ end ofthe target-recognizing siRNA sets the ruler for target RNA cleavage. Theposition is located between nucleotide 10 and 11 when counting inupstream direction from the target nucleotide paired to the 5′-mostnucleotide of the guide siRNA (see also FIG. 15A).

3.2.6 Sequence Effects and 2′-Deoxy Substitutions in the 3′ Overhang

A 2-nt 3′overhang is preferred for siRNA function. We wanted to know, ifthe sequence of the overhanging nucleotides contributes to targetrecognition, or if it is only a feature required for reconstitution ofthe endonuclease complex (RISC or siRNP). We synthesized sense andantisense siRNAs with AA, CC, GG, UU, and UG 3′ overhangs and includedthe 2′-deoxy modifications TdG and TT. The wild-type siRNAs contained AAin the sense 3′ overhang and UG in the antisense 3′ overhang (AA/UG).All siRNA duplexes were functional in the interference-assay and reducedtarget expression at least 5-fold (FIG. 17). The most efficient siRNAduplexes that reduced target expression more than 10-fold were of thesequence type NN/UG, NN/UU, NN/TdG, and NN/TT (N, any nucleotide). siRNAduplexes with an antisense siRNA 3′ overhang of AA, CC or GG were lessactive by a factor 2 to 4 when compared to the wild-type sequence UG orthe mutant UU. This reduction in RNAi efficiency is likely due to thecontribution of the penultimate 3′ nucleotide to sequence-specifictarget recognition, as the 3′ terminal nucleotide was changed from G toU without effect.

Changes in the sequence of the 3′ overhang of the sense siRNA did notreveal any sequence-dependent effects, which was expected, because thesense siRNA must not contribute to sense target mRNA recognition.

3.2.7 Sequence Specifity of Target Recognition

In order to examine the sequence-specifity of target-recognition, weintroduced sequence changes into the paired segments of siRNA duplexesand determined the efficiency of silencing. Sequence changes wereintroduced by inverting short segments of 3- or 4-nt length or as pointmutations (FIG. 18). The sequence changes in one siRNA strand werecompensated in the complementary siRNA strand to avoid pertubing thebase-paired siRNA duplex structure. The sequence of all 2-nt 3′overhangs was TT (T, 2′-deoxythymidine) to reduce costs of synthesis.The TT/TT reference siRNA duplex was comparable in RNAi to the wild-typesiRNA duplex AA/UG (FIG. 17). The ability to mediate reporter mRNAdestruction was quantified using the translation-based luminescenceassay. Duplexes of siRNAs with inverted sequence segments showeddramatically reduced ability for targeting the firefly luciferasereporter (FIG. 18). The sequence changes located between the 3′ end andthe middle of the antisense siRNA completely abolished target RNArecognition, but mutations near the 5′ end of the antisense siRNAexhibit a small degree of silencing. Transversion of the A/U base pairlocated directly opposite of the predicted target RNA to cleavage site,or one nucleotide further away from the predicted site, prevented targetRNA cleavage, therefore indicating that single mutation within thecentre of a siRNA duplex discriminate between mismatched targets.

3-3 Discussion

siRNAs are valuable reagents for inactivation of gene expression, notonly in insect cells, but also in mammalian cells, with a greatpotential for therapeutic application. We have systematically analysedthe structural determinants of siRNA duplexes required to promoteefficient target RNA degradation in D. melanogaster embryo lysate, thusproviding rules for the design of most potent siRNA duplexes. A perfectsiRNA duplex is able to silence gene expression with an efficiencycomparable to a 500 bp dsRNA, given that comparable quantities of totalRNA are used.

3.4 The siRNA User Guide

Efficiently silencing siRNA duplexes are preferably composed of 21-ntantisense siRNAs, and should be selected to form a 19 bp-double helixwith 2-nt 3′ overhanging ends. 2′-deoxy-substitutions of the 2-nt3′-over-hanging ribonucleotides do not affect RNAi, but help to reducethe costs of RNA synthesis and may enhance RNAse resistance of siRNAduplexes. More extensive 2′-deoxy or 2′-O-methyl modifications, however,reduce the ability of siRNAs to mediate RNAi, probably by interferingwith protein association for siRNAP assembly.

Target recognition is a highly sequence-specific process, mediated bythe siRNA complementary to the target. The 3′-most nucleotide of theguide siRNA does not contribute to specificity of target recognition,while the penultimate nucleotide of the 3′ overhang affects target RNAcleavage, and a mismatch reduces RNAi 2- to 4-fold. The 5′ end of aguide siRNA also appears more permissive for mismatched target RNArecognition when compared to the 3′ end. Nucleotides in the centre ofthe siRNA, located opposite the target RNA cleavage site, are importantspecificity determinants and even single nucleotide changes reduce RNAito undetectable level. This suggests that siRNA duplexes may be able todiscriminate mutant or polymorphic alleles in gene targetingexperiments, which may become an important feature for futuretherapeutic developments.

Sense and antisense siRNAs, when associated with the protein componentsof the endonuclease complex or its commitment complex, were suggested toplay distinct roles; the relative orientation of the siRNA duplex inthis complex defines which strand can be used for target recognition.Synthetic siRNA duplexes have dyad symmetry with respect to thedouble-helical structure, but not with respect to sequence. Theassociation of siRNA duplexes with the RNAi proteins in the D.melanogaster lysate will lead to formation of two asymmetric complexes.In such hypothetical complexes, the chiral environment is distinct forsense and antisense siRNA, hence their function. The predictionobviously does not apply to palindromic siRNA sequences, or to RNAiproteins that could associate as homodimers. To minimize sequenceeffects, which may affect the ratio of sense and antisense-targetingsiRNPs, we suggest to use siRNA sequences with identical 3′ overhangingsequences. We recommend to adjust the sequence of the overhang of thesense siRNA to that of the antisense 3′ overhang, because the sensesiRNA does not have a target in typical knockdown experiments. Asymmetryin reconstitution of sense and anti-sense-cleaving siRNPs could be(partially) responsible for the variation in RNAi efficiency observedfor various 21-nt siRNA duplexes with 2-nt 3′ overhangs used in thisstudy (FIG. 14). Alternatively, the nucleotide sequence at the targetsite and/or the accessibility of the target RNA structure may beresponsible for the variation in efficiency for these siRNA duplexes.

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1. Isolated double-stranded RNA molecule, wherein each RNA strand has alength from 19-25 nucleotides, wherein said RNA molecule is capable oftarget-specific nucleic acid modifications.
 2. The RNA molecule of claim1 wherein at least one strand has a 3′-overhang from 1-5 nucleotides. 3.The RNA molecule of claim 1 capable of target-specific RNA interferenceand/or DNA methylation.
 4. The RNA molecule of claim 1, wherein eachstrand has a length from 19-23, particularly from 20-22 nucleotides. 5.The RNA molecule of claim 2, wherein the 3′-overhang is from 1-3nucleotides.
 6. The RNA molecule of claim 2, wherein the 3′-overhang isstabilized against degradation.
 7. The RNA molecule of claim 1, whichcontains at least one modified nucleotide analogue.
 8. The RNA moleculeof claim 7, wherein the modified nucleotide analogue is selected fromsugar- or backbone modified ribonucleotides.
 9. The RNA moleculeaccording to claim 7, wherein the nucleotide analogue is asugar-modified ribonucleotide, wherein the 2′-OH group is replaced by agroup selected from H, OR, R, halo, SH, SR¹, NH₂, NHR, NR₂ or CN,wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.10. The RNA molecule of claim 7, wherein the nucleotide analogue is abackbone-modified ribonucleotide containing a phosphothioate group. 11.The RNA molecule of claim 1, which has a sequence having an identity ofat least 50 percent to a predetermined mRNA target molecule.
 12. The RNAmolecule of claim 11, wherein the identity is at least 70 percent.
 13. Amethod of preparing a-double-stranded RNA molecule of claim 1 comprisingthe steps: (a) synthesizing two RNA strands each having a length from19-25 nucleotides, wherein said RNA strands are capable of forming adouble-stranded RNA molecule, (b) combining the synthesized RNA strandsunder conditions, wherein a double-stranded RNA molecule is formed,which is capable of target-specific nucleic acid modifications.
 14. Themethod of claim 13, wherein the RNA strands are chemically synthesized.15. The method of claim 13, wherein the RNA strands are enzymaticallysynthesized.
 16. A method of mediating target-specific nucleic acidmodifications in a cell or an organism comprising the steps: (a)contacting said cell or organism with the double-stranded RNA moleculeof claim 1 under conditions wherein target-specific nucleic acidmodifications can occur, and (b) mediating a target-specific nucleicacid modification effected by the double-stranded RNA towards a targetnucleic acid having a sequence portion substantially corresponding tothe double-stranded RNA.
 17. The method of claim 16, wherein the nucleicacid modification is RNA interference and/or DNA methylation.
 18. Themethod of claim 16, wherein said contacting comprises introducing saiddouble-stranded RNA molecule into a target cell in which thetarget-specific nucleic acid modification can occur.
 19. The method ofclaim 18 wherein the introducing comprises a carrier-mediated deliveryor injection.
 20. Use of the method of claim 16 or determining thefunction of a gene in a cell or an organism.
 21. Use of the method ofclaim 16 for modulating the function of a gene in a cell or an organism.22. The use of claim 20, wherein the gene is associated with apathological condition.
 23. The use of claim 22, wherein the gene is apathogen-associated gene.
 24. The use of claim 23, wherein the gene is aviral gene.
 25. The use of claim 22, wherein the gene is atumor-associated gene.
 26. The use of claim 22, wherein the gene is anautoimmune disease-associated gene.
 27. Pharmaceutical compositioncontaining as an active agent at least one double-stranded RNA moleculeof claim 1 and a pharmaceutical carrier.
 28. The composition of claim 27for diagnostic applications.
 29. The composition of claim 27 fortherapeutic applications.